CN106282256B - A kind of biotransformation method producing cis- -4-Hydroxyproline - Google Patents
A kind of biotransformation method producing cis- -4-Hydroxyproline Download PDFInfo
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Abstract
The invention discloses a kind of biotransformation methods for producing cis- -4-Hydroxyproline.The method of the present invention by having co-expressed proline hydroxylase and proline transport protein e. coli bl21 (DE3) is intracellular, realize high yield it is cis- -4-Hydroxyproline efficiently synthesizing in recombinant bacterium.Compared with existing cis- -4-Hydroxyproline fermentation method, this method is easy to operate, sustainable, cost performance is high, has good industrial prospect.
Description
Technical field
The present invention relates to field of microbial fermentation, and in particular to a kind of biotransformation method for producing cis- -4-Hydroxyproline.
Background technique
Hydroxyproline (hydroxyproline), one of imino acid is usually upper with hydroxyl at the 4th, but sometimes
On the 3rd.Since there are two asymmetric carbon atoms, so there is 4 kinds of stereoisomers.Contain in animal glue and ossein
L- hydroxyproline.There is no D- hydroxyl dried meat acid in nature.It is the biologically active nitrogenous base being widely present in organism, it can
To be generated by proline hydroxylase.It has a wide range of applications in food, medicine and industrially.On cosmetics, hydroxyproline
Oxidant can be eliminated and adjust the effect of the redox state of cell, addition can skin care and anti-aging in cosmetics；
Dipalmitoyl hydroxyproline is generated using it as substrate condensation reaction, collagenous fibres contraction can be stimulated, high-efficient cleaning removes skin oxygen
Free radical；N- acetyl hydroxyproline has moisture-keeping function, and addition can effectively maintain the moisture of epidermal cell in cosmetics, makes skin
Keep elasticity；Medically, addition can prevent allergic inflammation in food and beverage, and derivative N- acetyl hydroxyproline is available
In treatment knot hoof tissue disease and rheumatic arthritis；Cis- -4-Hydroxyproline as the various cancers of cancer drug therapy, including
Liver, bladder, prostate, renal plevis etc..Ossein is prevented to be folded into a three stable helical conformations, to reduce tumor cell growth
With collagen over-deposit in fibrotic processes；Anti-hepatic fibrosis, anti-hypertension.
With rapid economic development, the trend of atmosphere pollution and global warming is worsening.The country mostly uses biological extraction
Method is hydrolyzed by protein such as gelatin, gelatine, casein, soybean epidermises with hydrochloric acid, with Nitrosation Process extract imidic acid after through resin
It refines, crystallize after chromatography, and the production of cis- -4-Hydroxyproline also needs to synthesize by chiral photo-isomerisation, bioanalysis closes
There is economics and ecological double meaning, bioanalysis synthesizing cis -4-Hydroxyproline gene work at cis- -4-Hydroxyproline
Journey bacterium mainly has Escherichia coli and yeast.
There are mainly two types of modes for bioanalysis synthesizing cis -4-Hydroxyproline at present: fermentation method and biotransformation method.Fermentation method
Raw material sources are extensive and renewable, at low cost, and yield is higher, and pollution is also smaller, but its regulation process is more complicated；Bioconversion
Method yield is high, and at low cost, process is simple, is conducive to downstream extraction operation.E. coli growth rates are fast, with short production cycle, and
Substrate is transported into cell membrane transport protein is needed to be transported into cell in Escherichia coli, has proline to turn in Escherichia coli at present
Albumen PutP is transported, overexpression is passed throughputpGene improves the proline of production bacterial strain in concentration of substrate intracellular, come improve it is cis--
The transformation efficiency of 4-Hydroxyproline, and substrate α-ketoglutaric acid is added leads to the serious shadow of acidity in reaction solution in initial reaction liquid
It rings reaction to carry out, needs to adjust pH to neutrality, however alpha Ketoglutarate sodium is used to replace alpha Ketoglutarate, pass through sodium alpha-ketoglutarate
Reaction is added using certain proportion as buffering with α-ketoglutaric acid and solves the problems, such as pH.This method can improve proline transport
It is slow to solve the problems, such as that the proline transport in production process enters cell velocity into cell velocity, and solves in reaction
The α-ketoglutaric acid of high concentration will affect pH value in reaction problem.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of biologies for producing cis- -4-Hydroxyproline
Conversion method, this method enter the speed of cell by changing proline, improve the yield of cis- -4-Hydroxyproline, save
PH step is adjusted, method is simplified.
A kind of biotransformation method producing cis- -4-Hydroxyproline is mentioned by accelerating the transport velocity of substrate proline
High-cis -4-Hydroxyproline yield.
In a kind of above-mentioned biotransformation method for producing cis- -4-Hydroxyproline, the transhipment speed for accelerating substrate proline
Rate, comprising the following steps: first building is overexpressed proline hydroxylase geneecp4hWith proline transport geneputpGene work
Journey bacterium；Fiber differentiation gene engineering bacteria obtains full cell again, is finally reacted with whole-cell catalytic and produces cis- -4-Hydroxyproline.
It is preferably as the above method, the proline hydroxylase geneecp4h, GenBank:FU758039.1；Dried meat
Propylhomoserin transporter geneputp Gene bankYP_003053684.1。
The above method the following steps are included:
Step 1, match seed culture medium, fermentation medium, reaction solution, soc culture medium, thallus washing lotion, and with culture dish, cone
Shape bottle, centrifuge tube sterilizing are spare；
Step 2, recombinant plasmid is constructed to obtain, and is transferred in host bacterial, then 12-14h is cultivated at 37 DEG C and obtains strain；
Step 3, seed culture medium, antibiotic and strain are sequentially ingressed into centrifuge tube, culture 9-10h obtains fermenting microbe；
Step 4, fermentation medium and fermenting microbe are accessed into conical flask, when shaking table culture to OD is at least 0.6, are added
To the IPTG of final concentration of 0.8-1.2mmol, after Fiber differentiation, supernatant is outwelled after being centrifuged 8-10min, washs filter with thallus washing lotion
It is spare that slag 2-4 obtains full cell after；
Step 5, full cell is added into conical flask after sterilizing, guarantees that the full dry cell weight being added is 0.41g/L, adds
Buffer solution and reaction solution carry out full cell effect；
Step 6, primary every 5-8h sampling, and product is detected by liquid phase, terminate when whole-cell catalytic 55-65h
Product cis -4-Hydroxyproline.
Be preferably as the above method, recombinant plasmid described in step 2 be pET28a-EP4H and pACYC-PutP,
PET28a-EP4H and pCDF-PutP or pET28a-EP4H-PutP.
It is preferably as the above method, condition of culture is 37 DEG C of temperature, revolving speed 250rpm in step 3.
It is preferably as the above method, the amount that fermenting microbe accesses in step 4 is 1-2%；The revolving speed of shaking table culture is
200rpm；The temperature of Fiber differentiation is 20 DEG C, revolving speed 200rpm；It is centrifuged 10min, centrifugal force 4000g/min。
It is preferably as the above method, the full cell effect time is 60h in step 5.
It is preferably as the above method, reaction solution is by 9-11g/L proline, 9-11g/L ketoglutaric acid, 9- in step 5
11g/L sodium alpha-ketoglutarate, ferrous sulfate and vitamin C mix, wherein ferrous sulfate and ascorbic molar concentration
Than for 3:4mmol/L.
Beneficial effect
Present approach provides a kind of biotransformation methods for producing cis- -4-Hydroxyproline.The method of the present invention by
E. coli bl21 (DE3) is intracellular to have co-expressed proline hydroxylase and proline transport protein, realizes the cis- -4- hydroxyl of high yield
Proline efficiently synthesizing in recombinant bacterium.Compared with existing cis- -4-Hydroxyproline fermentation method, this method is easy to operate,
It is sustainable, cost performance is high, have good industrial prospect.
Detailed description of the invention
Fig. 1 is recombinant plasmid pET28a-EP4H；
Fig. 2 is recombinant plasmid pACYC-PutP；
Fig. 3 is recombinant plasmid pCDF-PutP；
Fig. 4 is recombinant plasmid pET28a-EP4H-PutP.
Fig. 5 is the chromatogram that the embodiment of the present invention 4 produces cis- -4-Hydroxyproline, and wherein peak 1 is cis- -4-Hydroxyproline.
Fig. 6 is the chromatogram that the embodiment of the present invention 4 produces cis- -4-Hydroxyproline, and wherein peak 1 is proline.
Specific embodiment
The building of 1 recombinant plasmid of embodiment
Using genome of E.coli as template, withputpThe primer PCR at gene both ends expands, and obtainsputpSegment clone
To the site NdeI and XhoI of carrier pACYCDuet-1.Obtain recombinant plasmid pACYC-PutP.
The building of 2 recombinant plasmid of embodiment
Using genome of E.coli as template, withputpThe primer PCR at gene both ends expands, and obtainsputpSegment clone
To the site NdeI and XhoI of carrier pCDFDuet-1.Obtain recombinant plasmid pCDF-PutP.
The building of 3 recombinant plasmid of embodiment
Using genome of E.coli as template, withputpThe primer PCR at gene both ends expands, after the genetic fragment that amplifies
Digestion connection is implemented in body pACYC-duet after both ends are separately added into NdeI/XhoI restriction enzyme site.Restriction enzyme site is on carrier
NdeI/XhoI.Construct to obtain recombinant plasmid pACYC-PutP.PCR amplification makes the both ends gene PutP restriction enzyme site all be XhoI, after
PET28a-EP4H-PutP is constructed after connecting by digestion.
Embodiment 4
A kind of biotransformation method producing cis- -4-Hydroxyproline, comprising the following steps:
Step 1, with ferrous sulfate solution, seed culture medium, fermentation medium, reaction solution, soc culture medium, thallus washing lotion,
It is then spare with culture medium, conical flask, centrifuge tube sterilizing, in which:
Fermentation medium LB: peptone 10g/L, yeast powder 0.5 g/L of 0.5 g/L, NaCl, ampicillin 0.02%,
Chloramphenicol 0.01%, 0.01% pH7.0 of streptomysin.
Reaction solution: 10 g/L proline, 10 g/L α-ketoglutaric acids, 0.2mol/L potassium dihydrogen phosphate, 0.2mol/L
Dipotassium hydrogen phosphate, 0.8mmol/L vitamin C, 0.4 mmol/L ferrous sulfate.
Thallus washing lotion: 7.0 solution of PBS, 11.09g/LNaH2PO4 ,2.96g/LNa2HPO4。
Soc culture medium: peptone 4g/L, yeast powder 1g/L, NaCl 10mm/L, KCl 2.5mm/L, MgSO410mm/L,
MgCl220mm/L, 20 g/L of glucose.
The formula of seed culture medium is the same as fermentation medium LB.
Step 2, recombinant plasmid pET-28a-Ecp4H will be constructed to obtain in embodiment 1；PACYC- putP and bacterial strain pET-
28a-Ecp4H is transferred to Escherichia coli B21(DE3) after in 37 DEG C of incubator cultivate 12-14h obtain strain；
Step 3, the seed culture medium of 50ml centrifuge tube access 10ml and the antibiotic of 0.2um are taken, chooses on plate and a little connects
Enter to shake in pipe, 37 DEG C of revolving speeds of shaking table are to cultivate to obtain fermenting microbe under 250rpm；
Step 4, fermentation medium and fermenting microbe are accessed into conical flask, when shaking table culture to OD long is to 0.6, addition
To the IPTG of final concentration of 1mmol, 20 DEG C, be 4000 in centrifugal force after 200rpm induces 12hgIn the case where be centrifuged 10 points
Clock outwells supernatant, spare after using thallus washing lotion 2 times after collection thallus；
Step 5, the thallus of step 4 is added into conical flask after sterilizing, guarantees that the dry weight of the thallus after being added is 0.41g/
L, adds buffer solution and reaction solution carries out full cell effect, and reaction solution is by 9g/L proline, 9g/L ketone penta 2 in step 5
Acid, 9g/L alpha Ketoglutarate sodium, ferrous sulfate and vitamin C mix, wherein ferrous sulfate and ascorbic molar concentration
Than for 3:4mmol/L；
Step 6, a sample detection is taken every 6h, and product is detected by liquid phase, wait until that 55h-65h stops catalysis reaction,
Take final product.
Cis- -4-Hydroxyproline content, HPLC-ELSD analysis.
HPLC-ELSD analysis using evaporation photodetector, chromatographic column be the reversed column of Prevail C18 (250 mm ×
4.6 mm × 5 μm).HPLC condition are as follows: mobile phase A: contain 7 milliliters of trifluoroacetic acids, 0.653 milliliter of seven fluorine fourth in 1 liter of pure water
Acid, Mobile phase B: 100% acetonitrile, condition are as follows: 100% A；Flow velocity: 1.0 ml/min；Column temperature: 28.5 ± 1 °C；Sample volume: 10
μl.ELSD testing conditions: atomization temperature is 115 °C, gas velocity 3.2L/min.
Converted product analyzes (as shown in Figure 3) through HPLC-ELSD, and appearance time is 4.1 minutes and mark product appearance time one
It causes.
Embodiment 5
In addition to recombinant plasmid used in step 2 is pET-28a-Ecp4H and pCDF-PutP, other parts are the same as embodiment 4.
Embodiment 6
Except recombinant plasmid used in step 2 is pET-28a-Ecp4H-PutP, other parts are the same as embodiment 4.
Embodiment 7
Except in step 5 in reaction solution the volume ratio of alpha Ketoglutarate and alpha Ketoglutarate sodium salt be 1:5, other while implement
Example 6.
Comparative example
It is converted with above-mentioned production proline hydroxylase bacterial strain, and adjusts substrate α-ketoglutaric acid and sodium alpha-ketoglutarate
It is matched, other reaction conditions reach with embodiment 1, cis- in conversion fluid after conversion -4-Hydroxyproline maximum concentration
2.3g/L。
The yield that the method for the present invention produces cis- -4-Hydroxyproline is recorded in following table.
As can be seen that preparation method of the present invention turns proline by changing cell from the result of above-described embodiment
Capacity power improves the yield of cis- -4-Hydroxyproline, wherein the gene constructed with recombinant plasmid pET-28a-Ecp4H-PutP
Engineering bacteria effect more, in addition, the volume ratio of alpha Ketoglutarate and alpha Ketoglutarate sodium salt be 1:5 when, cis- -4-Hydroxyproline
Yield is also greatly improved, and therefore, the method for the present invention improves cis- -4- hydroxyl dried meat while simplifying experimentation
The yield of propylhomoserin has good market prospects.
Claims (7)
1. a kind of biotransformation method for producing cis- -4-Hydroxyproline, which is characterized in that by the transhipment for accelerating substrate proline
Rate improves the yield of cis- -4-Hydroxyproline；The transport velocity for accelerating substrate proline, comprising the following steps: first
Building is overexpressed the genetic engineering bacterium of proline hydroxylase gene ecp4h and proline transport gene putp；Induced medium again
Because engineering bacteria obtains full cell, is finally reacted with whole-cell catalytic and produce cis- -4-Hydroxyproline；The proline hydroxylase base
Because of Gene bank FU758039.1, proline transport gene Gene bankYP_003053684.1.
2. a kind of biotransformation method for producing cis- -4-Hydroxyproline according to claim 1, which is characterized in that including
Following steps:
Step 1, match seed culture medium, fermentation medium, reaction solution, soc culture medium, thallus washing lotion, and with culture dish, taper
Bottle, centrifuge tube sterilizing are spare；
Step 2, recombinant plasmid is constructed to obtain, and is transferred in host bacterial, then 12-14h is cultivated at 37 DEG C and obtains strain；
Step 3, seed culture medium, antibiotic and strain are sequentially ingressed into centrifuge tube, culture 9-10h obtains fermenting microbe；
Step 4, fermentation medium and fermenting microbe are accessed into conical flask, when shaking table culture to OD is at least 0.6, are added to end
Concentration is the IPTG of 0.8-1.2mmol, after Fiber differentiation, outwells supernatant after being centrifuged 8-10min, washs filter residue 2- with thallus washing lotion
It is spare that full cell is obtained after 4 times；
Step 5, full cell is added into conical flask after sterilizing, guarantees that the full dry cell weight being added is 0.41g/L, adds buffering
Solution and reaction solution carry out full cell effect；
Step 6, primary every 5-8h sampling, and product is detected by liquid phase, terminate to obtain product when whole-cell catalytic 55-65h
Cis- -4-Hydroxyproline.
3. a kind of biotransformation method for producing cis- -4-Hydroxyproline according to claim 2, which is characterized in that step 2
Described in recombinant plasmid be pET28a-EP4H and pACYC-PutP, pET28a-EP4H and pCDF-PutP or pET28a-EP4H-
PutP。
4. a kind of biotransformation method for producing cis- -4-Hydroxyproline according to claim 2, which is characterized in that step 3
Middle condition of culture is 37 DEG C of temperature, revolving speed 250rpm.
5. a kind of biotransformation method for producing cis- -4-Hydroxyproline according to claim 2, which is characterized in that step 4
The amount of middle fermenting microbe access is 1-2%；The revolving speed of shaking table culture is 200rpm；The temperature of Fiber differentiation is 20 DEG C, and revolving speed is
200rpm；It is centrifuged 10min, centrifugal speed 4000g/min.
6. a kind of biotransformation method for producing cis- -4-Hydroxyproline according to claim 2, which is characterized in that step 5
In the full cell effect time be 60h.
7. a kind of biotransformation method for producing cis- -4-Hydroxyproline according to claim 2, which is characterized in that step 5
Middle reaction solution is by 9-11g/L proline, 9-11g/L ketoglutaric acid, 9-11g/L sodium alpha-ketoglutarate, ferrous sulfate and vitamin
C is mixed, and wherein ferrous sulfate and ascorbic molar concentration rate are 3:4mmol/L.
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| CN104894152A (en) * | 2015-05-26 | 2015-09-09 | 江南大学 | Method for producing cis-4-hydroxy-L-proline through recombinant escherichia coli |
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| CN104894152A (en) * | 2015-05-26 | 2015-09-09 | 江南大学 | Method for producing cis-4-hydroxy-L-proline through recombinant escherichia coli |
Non-Patent Citations (3)
| Title |
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| Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1;Thi, MHB等;《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》;20130131;第97卷(第1期);247-257 |
| Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli;Falcioni, F等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20130531;第79卷(第9期);3091-3100 |
| 产顺式-4-L-羟脯氨酸工程菌的构建及转化条件的优化;赵利维等;《微生物学通报》;20160309;第43卷(第9期);1887-1894 |
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